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EP0451070B1 - Method for identifying or determining proteins and applications therefor - Google Patents

Method for identifying or determining proteins and applications therefor Download PDF

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Publication number
EP0451070B1
EP0451070B1 EP91430007A EP91430007A EP0451070B1 EP 0451070 B1 EP0451070 B1 EP 0451070B1 EP 91430007 A EP91430007 A EP 91430007A EP 91430007 A EP91430007 A EP 91430007A EP 0451070 B1 EP0451070 B1 EP 0451070B1
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EP
European Patent Office
Prior art keywords
peptide
protein
process according
amino acids
fragment
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EP91430007A
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German (de)
French (fr)
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EP0451070A1 (en
Inventor
Frédéric Jean
Jacques Barbet
Michel Delaage
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IMMUNOTECH SA
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Immunotech SAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/974—Aids related test
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials

Definitions

  • the present invention relates to a protein identification or assay method and its applications.
  • the precise immunological assay of a protein requires the use of antibodies whose affinity allows the detection of generally low amounts of antigen and whose specificity avoids interference with the other constituents of the medium in which this assay is carried out. Obtaining these antibodies frequently comes up against the difficulty of having a sufficiently abundant source and of suitable purity of the antigen studied.
  • WO-A-87 07957 describes a method for the assay by competition of a protein using an antibody directed against this protein, and a synthetic protein used as a tracer, also recognized by this antibody.
  • this method does not indicate that the synthetic protein can be used to produce antibodies, and moreover the establishment
  • This process requires the synthesis of a protein whose structure is very close to the natural protein against which the antibody is directed, so that the reactivity of this antibody is equivalent for the synthetic protein and the natural protein.
  • This process does not make it possible to choose the synthetic protein as a function of an enzymatic treatment of the sample capable of generating a peptide of structure identical to the synthetic protein.
  • EP-A-0 175 360 describes a method for assaying a protein using an antibody produced against a synthetic peptide whose corresponding epitope on the natural protein is exposed during the assay by the use of a weakly denaturing agent for the protein, which nevertheless preserves the binding capacity of the antibody.
  • This method does not indicate that the treatment of the sample could be an enzymatic proteolysis which cannot be considered as a partial denaturation of the protein.
  • the study of the primary structure of the protein to be assayed allows direct dosing, without subsequent modification or prior treatment, of specific proteolytic fragments. It differs from the works cited by three essential differences: the immunizing peptide is an artificial fragment, there is no extraction to be carried out to eliminate a natural fragment and there is no chemical modification to be carried out on the samples. .
  • the present application relates to a process for identifying or assaying proteins in which antibodies are prepared using a peptide made immunogenic if necessary, then using the antibodies thus obtained to assay the proteins desired from a sample containing them treated so as to release said peptide, characterized in that said peptide is selected from a known sequence of said protein as fragment or part of protein fragment releasable by enzymatic treatment then is prepared by synthesis.
  • Such a method has the advantages linked to the use of synthetic peptide immunogens: ease of preparation, choice of interesting epitope, and moreover makes it possible to avoid the problems of loss of affinity encountered in the case of a immunological assay of a native protein because, in this case, the immunogen corresponds perfectly to the antigen to be assayed, since for example it can be strictly identical to the antigen to be assayed. Furthermore, there is a wide range of enzymatic proteolytic cleavage techniques, the cleavage sites of which are specific and easily identifiable on the primary structure of the protein, allowing a wide choice in the selection of a peptide fragment.
  • the selection of said peptides can be made from a known amino acid sequence or deduced from the nucleic acid sequence of a corresponding gene.
  • peptides will correspond to a fragment of a protein such as a hormone or a viral protein.
  • the peptides will be selected because they correspond to proteolytic fragments which it will be possible to obtain by an enzymatic treatment such as tryptic fragments obtained after treatment of said protein with trypsin.
  • fragment part is meant a C-terminal, N-terminal or median part of the protein fragment.
  • fragment means fragment or part of fragment.
  • peptides will also be selected according to their size and their representativeness with respect to a single protein or with respect to a group of proteins.
  • one or more peptides for example 2, 3 or 4, can be used.
  • the peptide selected from a known sequence of proteins as a protein fragment which can be released by an appropriate treatment will contain from 6 to 30 amino acids and preferably from 6 to 20 amino acids.
  • peptides selected can thus be used as such according to the criteria defined above, it may however be advantageous to modify said peptides.
  • This modification can be done by substitution.
  • the number of amino acids being preserved, one or more of the amino acids of the selected peptide and preferably less than a quarter of the amino acids and in particular 1, 2 or 3 amino acids will be replaced by one or more different amino acids.
  • the substitute amino acid (s) will preferably have a free function, such as NH2, COOH, SH, for example.
  • amino acids which can replace one or more amino acids of the selected peptide mention may in particular be made of tyrosine or amino acids allowing oriented coupling to a carrier protein, an enzyme or any other molecule of interest.
  • amino acids to the selected peptide.
  • the amino acid (s) added will advantageously be chosen from those which can be used for substitutions.
  • the selected peptide can be found in the sequence of a protein but does not naturally exist in the state of peptide fragment.
  • the peptide can be selected for its specificity for a single protein. On the contrary, we can select the peptide for its specificity for a family of proteins.
  • the peptide selected may be in polymer form.
  • This polymer form can correspond, for example, to two fragments of proteins releasable by an appropriate treatment associated with one or more disulfide bridges via natural cysteines.
  • the polymerization may also be used for the purpose of modifying the immunogenic properties of one or more peptides.
  • the polymer may consist of identical or different monomers.
  • the monomers can be fixed, for example, by virtue of the amine, carboxylate or sulfhydryl functions, and in particular by means of one or more molecules joining these monomers.
  • the selection of the peptide can be done either because the protein considered is the only one to have said sequence, or on the contrary, because the sequence is common to a whole family of proteins and we want to assay the whole protein family.
  • the selected peptide fragment which allows for example to carry out a coupling to a carrier protein so as to orient the antigen-antibody response or to advantageously couple a molecule of interest such as an enzyme, or even to introduce an amino acid such as a tyrosine to effect labeling with iodine 125.
  • the technique of the present invention also eliminates a number of drawbacks from the techniques of the prior art such as the removal of troublesome proteins.
  • drawbacks from the techniques of the prior art such as the removal of troublesome proteins.
  • synthesis of the selected peptide is within the competence of a person skilled in the art and can be carried out according to well-known techniques among which mention may be made in particular of peptide synthesis in solid phase, according to Merrifield.
  • This method is particularly suitable for carrying out immunological assays of numerous protein hormones which are difficult to purify and which frequently have structural homologies.
  • Another interesting case is that of the immunological assay of a viral protein where three major difficulties may arise: the first is obtaining the antigen in sufficient quantity and purity to immunize animals, the second is the great antigenic variability that can be encountered through different strains of the same virus and the third is the interference caused by anti-viral protein antibodies present in the sera of infected people, which can mask all or part of the epitopes recognized by assay antibody. In this case, too, the determination of a proteolytic fragment makes it possible to resolve these various problems.
  • the choice of the trypsin fragmentation technique was chosen for its efficiency, low cost, simplicity and specificity (cleavage after the amino acids arginine and lysine).
  • the tryptic digestion map of the P25 protein of the reference strain HIV-1 BRU was established from the amino acid sequence extracted from the GENPRO database, using the BISANCE software (ISIC 2). Only the tryptic fragments having a size greater than or equal to six amino acids were selected. The selected fragments were then compared to the amino acid sequences of the P25 proteins of the other strains of HIV-1 and HIV-2 viruses contained in the NBRF and GENPRO databases.
  • This peptide is a complete tryptic fragment corresponding to amino acids 19 to 25 of the P25 protein of the reference strain HIV-1 BRU. It is strictly preserved across the different strains of HIV virus whose sequences have been studied.
  • This peptide corresponds to amino acids 101 to 110 of the NH2 end of the tryptic fragment comprising amino acids 101 to 131 for the reference strain HIV-1 BRU to which has been added in COOH an additional cysteine allowing chemical couplings oriented on the group SH.
  • This peptide is also strictly conserved across the different strains of HIV virus whose sequences have been studied.
  • This peptide is a complete tryptic fragment corresponding to amino acids 183 to 199 of the P25 protein of the reference strain HIV-1 BRU. It has a substitution of glutamic acid at position 187 with a glutamine for the HIV-2 ROD virus; a substitution of glutamic acid at position 187 with a glutamine a substitution of asparagine at position 193 with a serine and a deletion of alanine at position 194 with a glutamine for the HIV-2 SBLISY virus; a substitution of glutamic acid at position 187 with a glutamine and a substitution of valine at position 191 with an isoleucine for the HIV-2 FG virus; and a substitution of asparagine at position 183 for glycine for the HIV-1 Z2 virus.
  • This peptide corresponds to amino acids 210 to 227 of the COOH end of the tryptic fragment composed of amino acids 204 to 227 for the reference strain HIV-1 BRU. It has a substitution of methionine at position 215 with a leucine and a substitution of histidine at position 226 with a glutamine for the HIV-2 ROD virus; a substitution of methionine at position 215 with a leucine and a substitution of glycine at position 220 with an isoleucine for the HIV-2 virus SBLISY; substitution of methionine at position 215 with a leucine; a substitution of alanine at position 217 with a threonine and a substitution of histidine at position 226 with a glutamine for the HIV-2 FG virus; and a substitution of the glycine at position 225 with a serine for the viruses HIV-1 ELI, HIV-1 MAL and HIV-1 Z2.
  • the peptide N17K was coupled to bovine serum albumin via succinimidyl-4 (N-maleimido-methyl) cyclohexane 1-carboxylate (SMCC) which reacts on the one hand with the SH group of cysteine natural of the peptide and on the other hand with the epsilon-NH2 groups of the lysines of the carrier protein.
  • SMCC succinimidyl-4 (N-maleimido-methyl) cyclohexane 1-carboxylate
  • the different reagents are separated by molecular sieving on a column of Biogel® P2 (Bio-Rad®) balanced in 0.1M phosphate buffer, pH 6.
  • the peptide is then added to the solution of BSA-SMCC, in a molar ratio of 30/1 and the reaction mixture is incubated for 12 hours at 4 °.
  • the purification of the immunogen is carried out by molecular sieving on a column of Biogel® P4 (Bio-Rad®) balanced in PBS buffer, pH 7.2.
  • Four BALB / c mice were immunized at monthly intervals with 50 ⁇ g of this immunogen injected as a complete Freund's adjuvant, half by the subcutaneous route and half by the intraperitoneal route.
  • the antibodies thus obtained are characterized by their capacity to bind a radioactive tracer composed of the peptide N17K coupled to glycyl-L-tyrosine labeled with iodine 125.
  • a radioactive tracer composed of the peptide N17K coupled to glycyl-L-tyrosine labeled with iodine 125.
  • the glycyl-L-tyrosine conjugate SMCC (GT-SMCC) is then purified by reverse phase HPLC chromatography then coupled to the peptide N17K in solution at 0.5 mM in 0.1M phosphate buffer at pH 6.0 in a molar ratio of 1/1 . After 12 hours of incubation at 4 °, the N17K-SMCC-GT tracer is purified by reverse phase HPLC chromatography. The tracer N17K-SMCC-GT is labeled with iodine 125 by the chloramine T method and purified again by HPLC chromatography.
  • FIG. 1 An example of the result obtained with a mouse anti-serum immunized with the N17K-SMCC-BSA conjugate is presented in FIG. 1: to 100 ⁇ l of mouse anti-serum diluted to 1/1000 in PBS-BSA 0 buffer, 1%, pH 7.2, 50 ⁇ l of radiolabelled N17K-SMCC-GT tracer are added in the same buffer and 50 ⁇ l of N17K peptide or P25 HIV-1 BRU (Transgene) or P25 after treatment with trypsin, at different concentrations in normal human plasma. The antigen-antibody complexes are precipitated after an incubation of 12 hours at 4 ° by adding 200 ⁇ l of PEG 6000 at 20% in water and centrifugation. The radioactivity of the pellet is then measured. As shown in FIG. 1, the antibodies recognize both the N17K peptide and the P25 protein treated with trypsin, but not the native P25 protein.
  • the antibodies recognize both the N17K peptide and the P25 protein
  • the quantitative determination of the peptide N17K therefore makes it possible to carry out an assay of the P25 protein of the HIV-1 BRU virus but should also allow that of the P25 proteins of all the strains of HIV virus which have a structural homology with this fragment.
  • This peptide which corresponds to a tryptic fragment has no significant homology with all the protein sequences contained in the NBRF and GENPRO databases.
  • the immunizations were carried out with a peptide whose sequence is as follows: Glu-Leu-Thr-Cys-Gln-Arg, where a cysteine replaces the glycine present in the original fragment, in order to be able to couple this peptide to a carrier protein via the -SH group of this cysteine.
  • succinimidyl 4 N-maleimido-methyl cyclohexane-1 carboxylate (SMCC) which reacts, on the one hand with an SH group of a natural or supernumerary cysteine of the peptide and, on the other hand share, with the epsilon NH2 groups of the lysines of the carrier protein:
  • BSA bovine serum albumin
  • mice and two rabbits were immunized at monthly intervals with respectively 50 and 250 ⁇ g of this immunogen, injected subcutaneously as a complete Freund's adjuvant.
  • the assay is carried out by competition with an identical peptide coupled to glycyl-L-tyrosine via SMCC, radiolabelled with iodine 125 ( figure 2).

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Abstract

Method for identifying or determining proteins, in which antibodies are prepared with the aid of peptides which are made immunogenic if necessary and the antibodies thus obtained are then employed for determining the desired proteins from a sample containing them and treated so as to release the said peptides, in which the said peptide is selected from a known sequence of the said protein as a protein fragment which can be released by enzyme treatment and is then prepared by synthesis, and application to the identification or determination of proteins.

Description

La présente invention concerne un procédé d'identification ou de dosage de protéines et ses applications.The present invention relates to a protein identification or assay method and its applications.

Le dosage immunologique précis d'une protéine nécessite l'utilisation d'anticorps dont l'affinité permet la détection de quantités généralement faibles d'antigène et dont la spécificité évite les interférences avec les autres constituants du milieu dans lequel on effectue ce dosage. L'obtention de ces anticorps se heurte fréquemment à la difficulté de posséder une source suffisamment abondante et de pureté convenable de l'antigène étudié.The precise immunological assay of a protein requires the use of antibodies whose affinity allows the detection of generally low amounts of antigen and whose specificity avoids interference with the other constituents of the medium in which this assay is carried out. Obtaining these antibodies frequently comes up against the difficulty of having a sufficiently abundant source and of suitable purity of the antigen studied.

L'utilisation de peptides de synthèse comme immunogènes a permis d'obtenir des anticorps dirigés contre des antigènes rares ou dont on ne connaît que la séquence en acides aminés ou contre des épitopes particuliers, faiblement immunogéniques au sein d'une protéine. Cependant, dans la plupart des cas, les anticorps produits contre un peptide synthétique ne reconnaissent pas la protéine complète avec une affinité suffisante pour établir un dosage immunologique de sensibilité convenable.The use of synthetic peptides as immunogens has made it possible to obtain antibodies directed against rare antigens or of which only the amino acid sequence or against specific epitopes, weakly immunogenic within a protein, are known. However, in most cases, the antibodies produced against a synthetic peptide do not recognize the complete protein with sufficient affinity to establish an immunoassay of suitable sensitivity.

De nombreux traitements ont été envisagés pour éliminer les interférences dues aux autres constituants du milieu dans lequel on réalise un dosage immunologique d'une protéine. Une de ces méthodes consiste à effectuer le dosage après un traitement protéolytique de l'échantillon. Cette méthode, telle qu'elle est décrite, permet soit de détruire sélectivement les épitopes présents sur les protéines contaminantes en conservant ceux qui sont portés par la protéine à doser, soit de doser des fragments protéolytiques de la protéine gui portent des épitopes spécifiques, à l'aide d'anticorps obtenus par immunisation avec des fragments purifiés de la protéine clivée (demande de brevet européen N° 0.051.985). Cependant, chacun des deux aspects de cette méthodologie présente des inconvénients. La destruction sélective des épitopes portés par les protéines interférentes ne peut être réalisée que si l'on connaît précisément la localisation des épitopes reconnus par les anticorps du dosage et la nature des protéines contaminantes, ou par une étude extensive des différentes techniques de fragmentation protéolytique. En outre, le dosage de fragments protéolytiques à l'aide d'anticorps obtenus par immunisation avec les fragments purifiés nécessite une quantité importante de la protéine native très pure. De plus, la recherche d'anticorps spécifiques implique la caractérisation des fragments protéolytiques purifiés avant leur utilisation en temps qu'immunogène, ou l'étude approfondie de la spécificité des anticorps obtenus après immunisation avec les différents peptides.Many treatments have been envisaged to eliminate the interference due to the other constituents of the medium in which an immunological assay of a protein is carried out. One of these methods consists in carrying out the assay after a proteolytic treatment of the sample. This method, as described, makes it possible either to selectively destroy the epitopes present on the contaminating proteins while preserving those which are carried by the protein to be assayed, or to assay proteolytic fragments of the protein which carry specific epitopes, to using antibodies obtained by immunization with purified fragments of the cleaved protein (European patent application No. 0.051.985). However, each of the two aspects of this methodology has drawbacks. Selective destruction of epitopes carried by interfering proteins can only be achieved if we precisely know the location of the epitopes recognized by the antibodies in the assay and the nature of the contaminating proteins, or by an extensive study of the various proteolytic fragmentation techniques. In addition, the assay of proteolytic fragments using antibodies obtained by immunization with the purified fragments requires a large quantity of the very pure native protein. In addition, the search for specific antibodies involves the characterization of the purified proteolytic fragments before their use as an immunogen, or the in-depth study of the specificity of the antibodies obtained after immunization with the various peptides.

Cupo A., Rougon-Rapuzzi G. et Delaage M. (1981) dans Immunochemical detection of vasopressin precursors : artificial processing and quantification along the Hypothalamo-Hypophisial axis, Eur. J. Biochem., 115, 169-174 ont décrit une méthode de dosage du précurseur de la vasopressine au moyen d'anticorps dirigés contre la vasopressine elle-même, en reconstituant le déterminant antigénique avant le dosage par traitement enzymatique et chimique.Cupo A., Rougon-Rapuzzi G. and Delaage M. (1981) in Immunochemical detection of vasopressin precursors: artificial processing and quantification along the Hypothalamo-Hypophisial axis, Eur. J. Biochem., 115, 169-174 described a method for assaying the precursor of vasopressin by means of antibodies directed against the vasopressin itself, by reconstituting the antigenic determinant before the assay by enzymatic and chemical treatment.

Une approche équivalente a été également utilisée avec succès pour le dosage de la Met-enképhaline, en utilisant des anticorps produits contr un fragment naturel du précurseur. Ce dosage est réalisé après un traitement de l'échantillon par l'acide trichloracétique pour éliminer le fragment naturel, suivi d'un traitement par la trypsine (Cupo A., Pontarotti P.A., Jarry T. et Delaage M.A. (1984) dans "A new immunological approach to the detection and the quantitation of the Met-enkephalin precursors in rat brain", Neuropeptides, 4 : 375-387).An equivalent approach has also been successfully used for the assay of Met-enkephalin, using antibodies produced against a natural fragment of the precursor. This assay is carried out after treatment of the sample with trichloroacetic acid to remove the natural fragment, followed by treatment with trypsin (Cupo A., Pontarotti PA, Jarry T. and Delaage MA (1984) in "A new immunological approach to the detection and the quantitation of the Met-enkephalin precursors in rat brain ", Neuropeptides, 4: 375-387).

WO-A-87 07957 décrit un procédé de dosage par compétition d'une protéine à l'aide d'un anticorps dirigé contre cette protéine, et d'une protéine de synthèse utilisée comme traceur, également reconnue par cet anticorps. Toutefois, ce procédé n'indique pas que la protéine de synthèse puisse être utilisée pour produire des anticorps , et de plus la mise en oeuvre de ce procédé nécessite la synthèse d'une protéine dont la structure est très proche de la protéine naturelle contre laquelle est dirigé l'anticorps, afin que la réactivité de cet anticorps soit équivalente pour la protéine de synthèse et la protéine naturelle. Ce procédé ne permet pas de choisir la protéine de synthèse en fonction d'un traitement enzymatique de l'échantillon susceptible de générer un peptide de structure identique à la protéine de synthèse.WO-A-87 07957 describes a method for the assay by competition of a protein using an antibody directed against this protein, and a synthetic protein used as a tracer, also recognized by this antibody. However, this method does not indicate that the synthetic protein can be used to produce antibodies, and moreover the establishment This process requires the synthesis of a protein whose structure is very close to the natural protein against which the antibody is directed, so that the reactivity of this antibody is equivalent for the synthetic protein and the natural protein. This process does not make it possible to choose the synthetic protein as a function of an enzymatic treatment of the sample capable of generating a peptide of structure identical to the synthetic protein.

EP-A-0 175 360 décrit un procédé de dosage d'une protéine à l'aide d'un anticorps produit contre un peptide de synthèse dont l'épitope correspondant sur la protéine naturelle est exposé au cours du dosage par l'emploi d'un agent faiblement dénaturant pour la protéine, qui préserve néanmoins la capacité de liaison de l'anticorps.EP-A-0 175 360 describes a method for assaying a protein using an antibody produced against a synthetic peptide whose corresponding epitope on the natural protein is exposed during the assay by the use of a weakly denaturing agent for the protein, which nevertheless preserves the binding capacity of the antibody.

Ce procédé n'indique pas que le traitement de l'échantillon pourrait être une protéolyse enzymatique qui ne peut être considérée comme une dénaturation partielle de la protéine.This method does not indicate that the treatment of the sample could be an enzymatic proteolysis which cannot be considered as a partial denaturation of the protein.

Il est impossible de choisir à l'avance une séquence en acide aminés correspondant à un épitope qui aurait une chance d'être exposé par ce traitement.It is impossible to choose in advance an amino acid sequence corresponding to an epitope that would have a chance of being exposed by this treatment.

Plus simplement, dans la présente invention, l'étude de la structure primaire de la protéine à doser permet le dosage direct et sans modification ultérieure ou traitement préalable, de fragments protéolytiques spécifiques. Elle se distingue des travaux cités par trois différences essentielles : le peptide immunisant est un fragment artificiel, il n'y a pas d'extraction à réaliser pour éliminer un fragment naturel et il n'y a pas de modification chimique à opérer sur les échantillons.More simply, in the present invention, the study of the primary structure of the protein to be assayed allows direct dosing, without subsequent modification or prior treatment, of specific proteolytic fragments. It differs from the works cited by three essential differences: the immunizing peptide is an artificial fragment, there is no extraction to be carried out to eliminate a natural fragment and there is no chemical modification to be carried out on the samples. .

C'est pourquoi la présente demande a pour objet un procédé d'identification ou de dosage de protéines dans lequel l'on prépare des anticorps à l'aide d'un peptide rendu si nécessaire immunogène, puis utilise les anticorps ainsi obtenus pour doser les protéines désirées à partir d'un échantillon les renfermant traité de manière à libérer ledit peptide, caractérisé en ce que ledit peptide est sélectionné à partir d'une séquence connue de ladite protéine en tant que fragment ou partie de fragment de protéine libérable par traitement enzymatique puis est préparé par synthèse.This is why the present application relates to a process for identifying or assaying proteins in which antibodies are prepared using a peptide made immunogenic if necessary, then using the antibodies thus obtained to assay the proteins desired from a sample containing them treated so as to release said peptide, characterized in that said peptide is selected from a known sequence of said protein as fragment or part of protein fragment releasable by enzymatic treatment then is prepared by synthesis.

Un tel procédé possède les avantages liés à l'utilisation d'immunogènes peptidiques de synthèse : facilité de préparation, choix de l'épitope intéressant, et permet de plus d'éviter les problèmes de perte d'affinité rencontrés dans le cas d'un dosage immunologique d'une protéine native car, dans ce cas, l'immunogène correspond parfaitement à l'antigène à doser, puisque par exemple il peut être strictement identique à l'antigène à doser. Par ailleurs, il existe un large éventail de techniques de clivage protéolytique enzymatique, dont les sites de coupure sont spécifiques et facilement identifiables sur la structure primaire de la protéine, permettant un large choix dans la sélection d'un fragment peptidique.Such a method has the advantages linked to the use of synthetic peptide immunogens: ease of preparation, choice of interesting epitope, and moreover makes it possible to avoid the problems of loss of affinity encountered in the case of a immunological assay of a native protein because, in this case, the immunogen corresponds perfectly to the antigen to be assayed, since for example it can be strictly identical to the antigen to be assayed. Furthermore, there is a wide range of enzymatic proteolytic cleavage techniques, the cleavage sites of which are specific and easily identifiable on the primary structure of the protein, allowing a wide choice in the selection of a peptide fragment.

La préparation d'anticorps à l'aide de peptides est bien connue de l'état de la technique ; dans le cas ou lesdits peptides ne sont pas ou sont insuffisamment immunogènes, il est possible de leur conférer ces propriétés, par exemple en fixant lesdits peptides sur une macromolécule, notamment d'origine protéique.The preparation of antibodies using peptides is well known in the art; in the case where said peptides are not or are insufficiently immunogenic, it is possible to confer these properties on them, for example by fixing said peptides on a macromolecule, in particular of protein origin.

La sélection desdits peptides pourra se faire à partir d'une séquence connue en acides aminés ou déduite de la séquence en acides nucléiques d'un gène correspondant.The selection of said peptides can be made from a known amino acid sequence or deduced from the nucleic acid sequence of a corresponding gene.

Ces peptides correspondront à un fragment d'une protéine telle qu'une hormone ou une protéine virale.These peptides will correspond to a fragment of a protein such as a hormone or a viral protein.

Les peptides seront sélectionnés parce qu'ils correspondent à des fragments protéolytiques qu'il sera possible d'obtenir par un traitement enzymatique tels que des fragments trypsiques obtenus après traitement de ladite protéine par la trypsine.The peptides will be selected because they correspond to proteolytic fragments which it will be possible to obtain by an enzymatic treatment such as tryptic fragments obtained after treatment of said protein with trypsin.

Par "partie de fragment", l'on entend une partie C-terminale, N-terminale ou médiane du fragment protéique.By "fragment part" is meant a C-terminal, N-terminal or median part of the protein fragment.

Dans ce qui suit, par "fragment", l'on entend fragment ou partie de fragment.In what follows, by "fragment" means fragment or part of fragment.

Il est bien sûr possible d'utiliser d'autres traitements enzymatiques simples ou associant plusieurs enzymes, telles que par exemple trypsine, chymotrypsine, proline endopeptidase.It is of course possible to use other simple enzymatic treatments or those combining several enzymes, such as for example trypsin, chymotrypsin, proline endopeptidase.

Ces peptides seront également sélectionnés en fonction de leur taille et de leur représentativité par rapport à une protéine unique ou par rapport à un groupe de protéines.These peptides will also be selected according to their size and their representativeness with respect to a single protein or with respect to a group of proteins.

En vue de la mise en oeuvre du procédé selon la présente invention, on peut utiliser un ou plusieurs peptides, par exemple 2, 3 ou 4.In order to carry out the process according to the present invention, one or more peptides, for example 2, 3 or 4, can be used.

Dans des conditions préférentielles de mise en oeuvre du procédé ci-dessus décrit, le peptide sélectionné à partir d'une séquence connue de protéines en tant que fragment de protéine libérable par un traitement approprié comportera de 6 à 30 acides aminés et de préférence de 6 à 20 acides aminés.Under preferential conditions for carrying out the process described above, the peptide selected from a known sequence of proteins as a protein fragment which can be released by an appropriate treatment will contain from 6 to 30 amino acids and preferably from 6 to 20 amino acids.

Si les peptides sélectionnés peuvent ainsi être utilisés tels quels en fonction des critères ci-dessus définis, il peut être toutefois avantageux de modifier lesdits peptides.If the peptides selected can thus be used as such according to the criteria defined above, it may however be advantageous to modify said peptides.

Cette modification pourra se faire par substitution. Ainsi, le nombre d'acides aminés étant conservé, un ou plusieurs des acides aminés du peptide sélectionné et de préférence moins du quart des acides aminés et notamment 1, 2 ou 3 acides aminés seront remplacés par un ou plusieurs acides aminés différents. Le ou les acide(s) aminé(s) de substitution comporteront de préférence une fonction libre, telle que NH₂, COOH, SH, par exemple.This modification can be done by substitution. Thus, the number of amino acids being preserved, one or more of the amino acids of the selected peptide and preferably less than a quarter of the amino acids and in particular 1, 2 or 3 amino acids will be replaced by one or more different amino acids. The substitute amino acid (s) will preferably have a free function, such as NH₂, COOH, SH, for example.

Comme acides aminés pouvant substituer un ou plusieurs acides aminés du peptide sélectionné, on peut citer notamment la tyrosine ou les acides aminés permettant un couplage orienté sur une protéine porteuse, une enzyme ou toute autre molécule intéressante.As amino acids which can replace one or more amino acids of the selected peptide, mention may in particular be made of tyrosine or amino acids allowing oriented coupling to a carrier protein, an enzyme or any other molecule of interest.

Il est également intéressant d'ajouter un ou plusieurs acides aminés au peptide sélectionné. Le ou les acide(s) aminé(s) ajoutés seront avantageusement choisis parmi ceux pouvant être utilisés pour les substitutions.It is also interesting to add one or more amino acids to the selected peptide. The amino acid (s) added will advantageously be chosen from those which can be used for substitutions.

Il peut également être intéressant de modifier ledit fragment par délétion d'un ou plusieurs acides aminés, de préférence moins de la moitié des acides aminés.It may also be advantageous to modify said fragment by deletion of one or more amino acids, preferably less than half of the amino acids.

Afin d'éviter des interférences lors de l'identification ou du dosage des protéines selon la présente invention, le peptide sélectionné peut être retrouvé dans la séquence d'une protéine mais n'existe pas naturellement à l'état de fragment peptidique.In order to avoid interference during the identification or the assay of the proteins according to the present invention, the selected peptide can be found in the sequence of a protein but does not naturally exist in the state of peptide fragment.

Comme déjà indiqué, le peptide pourra être sélectionné pour sa spécificité pour une protéine unique. Au contraire, on pourra sélectionner le peptide pour sa spécificité pour une famille de protéines.As already indicated, the peptide can be selected for its specificity for a single protein. On the contrary, we can select the peptide for its specificity for a family of proteins.

En vue de la mise en oeuvre du procédé ci-dessus décrit, le peptide sélectionné pourra se trouver sous forme polymère. Cette forme polymère peut correspondre par exemple à deux fragments de protéines libérables par un traitement approprié associés par un ou plusieurs ponts disulfures par l'intermédiaire de cystéines naturelles.With a view to implementing the process described above, the peptide selected may be in polymer form. This polymer form can correspond, for example, to two fragments of proteins releasable by an appropriate treatment associated with one or more disulfide bridges via natural cysteines.

La polymérisation pourra être également utilisée dans le but de modifier les propriétés immunogéniques d'un ou plusieurs peptides. Le polymère pourra être constitué de monomères identiques ou non. La fixation des monomères pourra se faire, par exemple, grâce aux fonctions amines, carboxylates ou sulfhydryles, et notamment par l'intermédiaire d'une ou plusieurs molécules joignant ces monomères.The polymerization may also be used for the purpose of modifying the immunogenic properties of one or more peptides. The polymer may consist of identical or different monomers. The monomers can be fixed, for example, by virtue of the amine, carboxylate or sulfhydryl functions, and in particular by means of one or more molecules joining these monomers.

Les avantages du procédé ci-dessus décrit sont nombreux.The advantages of the method described above are numerous.

Il n'y a aucun besoin de détenir physiquement la protéine. Une connaissance bibliographique de la séquence ou d'une partie de la séquence de la ou des protéine(s) à doser suffit. L'homme de l'art, connaissant les techniques de clivage enzymatique grâce à une ou plusieurs enzymes pourra déterminer quels peptides peuvent correspondre à un fragment libérable par traitement approprié.There is no need to physically hold the protein. Bibliographic knowledge of the sequence or part of the sequence of the protein (s) to be assayed is sufficient. Those skilled in the art, knowing the techniques for enzymatic cleavage using one or more enzymes, will be able to determine which peptides can correspond to a releasable fragment by appropriate treatment.

La sélection du peptide pourra se faire soit parce que la protéine considérée est la seule à posséder ladite séquence, soit au contraire, parce que la séquence est commune à toute une famille de protéines et que l'on veut doser la famille de protéine entière.The selection of the peptide can be done either because the protein considered is the only one to have said sequence, or on the contrary, because the sequence is common to a whole family of proteins and we want to assay the whole protein family.

Il est également possible de sélectionner a priori une séquence vis-à-vis de critères particuliers tels que le critère de taille du peptide.It is also possible to select a priori a sequence with regard to particular criteria such as the peptide size criterion.

Il est possible de modifier le fragment peptidique sélectionné, ce qui permet par exemple de réaliser un couplage à une protéine porteuse de façon à orienter la réponse antigène-anticorps ou de coupler avantageusement une molécule d'intérêt telle qu'une enzyme, ou encore à introduire un acide aminé tel qu'une tyrosine pour effectuer un marquage par l'iode 125.It is possible to modify the selected peptide fragment, which allows for example to carry out a coupling to a carrier protein so as to orient the antigen-antibody response or to advantageously couple a molecule of interest such as an enzyme, or even to introduce an amino acid such as a tyrosine to effect labeling with iodine 125.

La technique de la présente invention supprime aussi nombre d'inconvénients des techniques de l'art antérieur tels que l'élimination des protéines gênantes. En effet, dans les techniques de l'art antérieur, il est difficile de trouver une procédure efficace et de longs efforts sont nécessaires pour déterminer la technique de clivage enzymatique et/ou chimique permettant l'élimination des protéines parasites.The technique of the present invention also eliminates a number of drawbacks from the techniques of the prior art such as the removal of troublesome proteins. In Indeed, in the techniques of the prior art, it is difficult to find an effective procedure and long efforts are necessary to determine the enzymatic and / or chemical cleavage technique allowing the elimination of the parasitic proteins.

La synthèse du peptide sélectionné est de la compétence de l'homme de l'art et peut être réalisée selon les techniques bien connues parmi lesquelles on peut citer notamment la synthèse peptidique en phase solide, selon Merrifield.The synthesis of the selected peptide is within the competence of a person skilled in the art and can be carried out according to well-known techniques among which mention may be made in particular of peptide synthesis in solid phase, according to Merrifield.

Ce procédé est particulièrement adapté à la réalisation de dosages immunologiques de nombreuses hormones protéiques difficiles à purifier et présentant fréquemment des homologies de structure. Un autre cas intéressant est celui du dosage immunologique d'une protéine virale où trois difficultés majeures peuvent se présenter : la première est l'obtention de l'antigène en quantité et en pureté suffisante pour immuniser des animaux, la deuxième est la grande variabilité antigénique que l'on peut rencontrer à travers les différentes souches d'un même virus et la troisième est l'interférence provoquée par les anticorps anti-protéines virales présents dans les sérums des personnes infectées, qui peuvent masquer tout ou partie des épitopes reconnus par les anticorps du dosage. Dans ce cas, également, le dosage d'un fragment protéolytique permet de résoudre ces différents problèmes. En effet, l'utilisation d'un peptide de synthèse correspondant à un fragment protéolytique permet d'avoir un immunogène très pur et en quantité importante ; le choix judicieux de la séquence permet de sélectionner des fragments conservés au sein des différentes souches d'un même virus ou au contraire spécifique d'une souche et les épitopes des anticorps du dosage n'existant pas à l'état naturel, ne peuvent être masqués par les anticorps sériques.This method is particularly suitable for carrying out immunological assays of numerous protein hormones which are difficult to purify and which frequently have structural homologies. Another interesting case is that of the immunological assay of a viral protein where three major difficulties may arise: the first is obtaining the antigen in sufficient quantity and purity to immunize animals, the second is the great antigenic variability that can be encountered through different strains of the same virus and the third is the interference caused by anti-viral protein antibodies present in the sera of infected people, which can mask all or part of the epitopes recognized by assay antibody. In this case, too, the determination of a proteolytic fragment makes it possible to resolve these various problems. Indeed, the use of a synthetic peptide corresponding to a proteolytic fragment makes it possible to have a very pure immunogen and in large quantity; the judicious choice of the sequence makes it possible to select fragments conserved within the different strains of the same virus or on the contrary specific for a strain and the epitopes of the antibodies of the assay not existing in their natural state, cannot be masked by serum antibodies.

Les exemples qui suivent illustrent la présente invention sans toutefois la limiter.The examples which follow illustrate the present invention without however limiting it.

Exemple 1 : Dosage de la protéine P25 du virus HIV EXAMPLE 1 Assay of the P25 Protein of the HIV Virus 1. Sélection des fragments peptidiques 1. Selection of peptide fragments

Le choix de la technique de fragmentation par la trypsine a été retenu pour son efficacité, son faible coût, sa simplicité et sa spécificité (clivage après les acides aminés arginine et lysine). La carte de digestion trypsique de la protéine P25 de la souche de référence HIV-1 BRU a été établie à partir de la séquence en acides aminés extraite de la banque de données GENPRO, à l'aide du logiciel BISANCE (CITI 2). Seuls les fragments trypsiques ayant une taille supérieure ou égale à six acides aminés ont été sélectionnés. Les fragments retenus ont été ensuite comparés aux séquences en acides aminés des protéines P25 des autres souches de virus HIV-1 et HIV-2 contenues dans les banques de données NBRF et GENPRO. Seuls les fragments trypsiques, ou les peptides de plus de six acides aminés correspondant à une extrémité COOH ou NH₂ d'un fragment trypsique, qui présentaient plus de 80 % d'homologie de séquence à travers les différentes souches de virus HIV-1 et HIV-2 ont été conservés. Ces peptides ont été ensuite comparés à l'ensemble des séquences protéiques contenues dans les banques de données NBRF et GENPRO et aucune homologie significative avec des séquences autres que celles des protéines P25 des virus HIV et des virus SIV n'a été relevée.The choice of the trypsin fragmentation technique was chosen for its efficiency, low cost, simplicity and specificity (cleavage after the amino acids arginine and lysine). The tryptic digestion map of the P25 protein of the reference strain HIV-1 BRU was established from the amino acid sequence extracted from the GENPRO database, using the BISANCE software (ISIC 2). Only the tryptic fragments having a size greater than or equal to six amino acids were selected. The selected fragments were then compared to the amino acid sequences of the P25 proteins of the other strains of HIV-1 and HIV-2 viruses contained in the NBRF and GENPRO databases. Only the tryptic fragments, or the peptides of more than six amino acids corresponding to a COOH or NH₂ end of a tryptic fragment, which exhibited more than 80% of sequence homology across the different strains of HIV-1 and HIV virus -2 have been retained. These peptides were then compared with all the protein sequences contained in the NBRF and GENPRO databases and no significant homology with sequences other than those of the P25 proteins of HIV viruses and SIV viruses was noted.

Quatre peptides ont été sélectionnés suivant les critères précédents et synthétisés :Four peptides were selected according to the previous criteria and synthesized:

Le peptide T7KThe T7K peptide

Thr-Leu-Asn-Ala-Trp-Val-Lys
   Ce peptide est un fragment trypsique complet correspondant aux acides aminés 19 à 25 de la protéine P25 de la souche de référence HIV-1 BRU. Il est strictement conservé à travers les différentes souches de virus HIV dont les séquences ont été étudiées.Thr-Leu-Asn-Ala-Trp-Val-Lys
This peptide is a complete tryptic fragment corresponding to amino acids 19 to 25 of the P25 protein of the reference strain HIV-1 BRU. It is strictly preserved across the different strains of HIV virus whose sequences have been studied.

Le peptide G11CThe G11C peptide

Gly-Ser-Asp-Ile-Ala-Gly-Thr-Thr-Ser-Thr-Cys
   Ce peptide correspond aux acides aminés 101 à 110 de l'extrémité NH2 du fragment trypsique comprenant les acides aminés 101 à 131 pour la souche de référence HIV-1 BRU auxquels a été rajouté en COOH une cystéine supplémentaire permettant des couplages chimiques orientés sur le groupement SH. Ce peptide est également strictement conservé à travers les différentes souches de virus HIV dont les séquences ont été étudiées.Gly-Ser-Asp-Ile-Ala-Gly-Thr-Thr-Ser-Thr-Cys
This peptide corresponds to amino acids 101 to 110 of the NH2 end of the tryptic fragment comprising amino acids 101 to 131 for the reference strain HIV-1 BRU to which has been added in COOH an additional cysteine allowing chemical couplings oriented on the group SH. This peptide is also strictly conserved across the different strains of HIV virus whose sequences have been studied.

Le peptide N17KThe N17K peptide

Figure imgb0001

   Ce peptide est un fragment trypsique complet correspondant aux acides aminés 183 à 199 de la protéine P25 de la souche de référence HIV-1 BRU. Il présente une substitution de l'acide glutamique en position 187 par une glutamine pour le virus HIV-2 ROD ; une substitution de l'acide glutamique en position 187 par une glutamine une substitution de l'asparagine en position 193 par une sérine et une délétion de l'alanine en position 194 par une glutamine pour le virus HIV-2 SBLISY ; une substitution de l'acide glutamique en position 187 par une glutamine et une substitution de la valine en position 191 par une isoleucine pour le virus HIV-2 FG ; et une substitution de l'asparagine en position 183 en glycine pour le virus HIV-1 Z2.
Figure imgb0001

This peptide is a complete tryptic fragment corresponding to amino acids 183 to 199 of the P25 protein of the reference strain HIV-1 BRU. It has a substitution of glutamic acid at position 187 with a glutamine for the HIV-2 ROD virus; a substitution of glutamic acid at position 187 with a glutamine a substitution of asparagine at position 193 with a serine and a deletion of alanine at position 194 with a glutamine for the HIV-2 SBLISY virus; a substitution of glutamic acid at position 187 with a glutamine and a substitution of valine at position 191 with an isoleucine for the HIV-2 FG virus; and a substitution of asparagine at position 183 for glycine for the HIV-1 Z2 virus.

Le peptide T18KThe T18K peptide

Figure imgb0002

   Ce peptide correspond aux acides aminés 210 à 227 de l'extrémité COOH du fragment trypsique composé des acides aminés 204 à 227 pour la souche de référence HIV-1 BRU. Il présente une substitution de la méthionine en position 215 par une leucine et une substitution de l'histidine en position 226 par une glutamine pour le virus HIV-2 ROD; une substitution de la méthionine en position 215 par une leucine et une substitution de la glycine en position 220 par une isoleucine pour le virus HIV-2 SBLISY; une substitution de la méthionine en position 215 par une leucine; une substitution de l'alanine en position 217 par une thréonine et une substitution de l'histidine en position 226 par une glutamine pour le virus HIV-2 FG ; et une substitution de la glycine en position 225 par une sérine pour les virus HIV-1 ELI, HIV-1 MAL et HIV-1 Z2.
Figure imgb0002

This peptide corresponds to amino acids 210 to 227 of the COOH end of the tryptic fragment composed of amino acids 204 to 227 for the reference strain HIV-1 BRU. It has a substitution of methionine at position 215 with a leucine and a substitution of histidine at position 226 with a glutamine for the HIV-2 ROD virus; a substitution of methionine at position 215 with a leucine and a substitution of glycine at position 220 with an isoleucine for the HIV-2 virus SBLISY; substitution of methionine at position 215 with a leucine; a substitution of alanine at position 217 with a threonine and a substitution of histidine at position 226 with a glutamine for the HIV-2 FG virus; and a substitution of the glycine at position 225 with a serine for the viruses HIV-1 ELI, HIV-1 MAL and HIV-1 Z2.

2. Production d'anticorps 2. Antibody production

Ces quatre peptides ont été couplés à différentes protéines porteuses afin d'être utilisés comme immunogènes.These four peptides have been coupled to different carrier proteins for use as immunogens.

En particulier, le peptide N17K a été couplé à de la sérum albumine bovine par l'intermédiaire de succinimidyl-4 (N-maléimido-méthyl) cyclohexane 1-carboxylate (SMCC) qui réagit d'une part avec le groupement SH de la cystéine naturelle du peptide et d'autre part avec les groupements epsilon-NH2 des lysines de la protéine porteuse. A de la sérum albumine bovine (Serva) en solution à 5 mg/ml dans un tampon phosphate 0.1M, pH 7, on ajoute du SMCC (Pierce) en solution à 25 mg/ml dans le DMF dans un rapport molaire de 1/50. Après une incubation d'une heure à température ambiante, les différents réactifs sont séparés par tamisage moléculaire sur une colonne de Biogel® P2 (Bio-Rad®) équilibrée en tampon phosphate 0.1M, pH 6. Le peptide est ensuite ajouté à la solution de BSA-SMCC, dans un rapport molaire de 30/1 et le mélange réactionnel est incubé 12 heures à 4°. La purification de l'immunogène est réalisée par tamisage moléculaire sur une colonne de Biogel® P4 (Bio-Rad®) équilibrée en tampon PBS, pH 7,2. Quatre souris BALB/c ont été immunisées à intervalle mensuel avec 50 µg de cet immunogène injecté en adjuvant complet de Freund, pour moitié par voie sous cutanée et pour moitié par voie intrapéritonéale.In particular, the peptide N17K was coupled to bovine serum albumin via succinimidyl-4 (N-maleimido-methyl) cyclohexane 1-carboxylate (SMCC) which reacts on the one hand with the SH group of cysteine natural of the peptide and on the other hand with the epsilon-NH2 groups of the lysines of the carrier protein. To bovine serum albumin (Serva) in solution at 5 mg / ml in 0.1M phosphate buffer, pH 7, SMCC (Pierce) is added in solution at 25 mg / ml in DMF in a molar ratio of 1 / 50. After an incubation of one hour at room temperature, the different reagents are separated by molecular sieving on a column of Biogel® P2 (Bio-Rad®) balanced in 0.1M phosphate buffer, pH 6. The peptide is then added to the solution of BSA-SMCC, in a molar ratio of 30/1 and the reaction mixture is incubated for 12 hours at 4 °. The purification of the immunogen is carried out by molecular sieving on a column of Biogel® P4 (Bio-Rad®) balanced in PBS buffer, pH 7.2. Four BALB / c mice were immunized at monthly intervals with 50 μg of this immunogen injected as a complete Freund's adjuvant, half by the subcutaneous route and half by the intraperitoneal route.

3. Caractérisation des anticorps 3. Characterization of antibodies

Les anticorps ainsi obtenus sont caractérisés par leur capacité à lier un traceur radioactif composé du peptide N17K couplé à glycyl-L-tyrosine marqué à l'iode 125. Dans un premier temps, à une solution de glycyl-L-tyrosine (Sigma) 20 mM en tampon phosphate 0.1M à pH 7,0, on ajoute du SMCC (Pierce) en solution à 25 mg/ml dans le DMF dans un rapport molaire de 1/1 et le mélange réactionnel est incubé 2 heures à 20°. Le conjugué glycyl-L-tyrosine SMCC (GT-SMCC) est alors purifié par chromatographie HPLC en phase reverse puis couplé au peptide N17K en solution à 0.5 mM en tampon phosphate 0.1M à pH 6,0 dans un rapport molaire de 1/1. Après 12 heures d'incubation à 4°, le traceur N17K-SMCC-GT est purifié par chromatographie HPLC en phase reverse. Le traceur N17K-SMCC-GT est marqué à l'iode 125 par la méthode à la chloramine T et purifié à nouveau par chromatographie HPLC.The antibodies thus obtained are characterized by their capacity to bind a radioactive tracer composed of the peptide N17K coupled to glycyl-L-tyrosine labeled with iodine 125. Firstly, to a solution of glycyl-L-tyrosine (Sigma) 20 mM in 0.1M phosphate buffer at pH 7.0, SMCC (Pierce) is added in solution at 25 mg / ml in DMF in a molar ratio of 1/1 and the reaction mixture is incubated for 2 hours at 20 °. The glycyl-L-tyrosine conjugate SMCC (GT-SMCC) is then purified by reverse phase HPLC chromatography then coupled to the peptide N17K in solution at 0.5 mM in 0.1M phosphate buffer at pH 6.0 in a molar ratio of 1/1 . After 12 hours of incubation at 4 °, the N17K-SMCC-GT tracer is purified by reverse phase HPLC chromatography. The tracer N17K-SMCC-GT is labeled with iodine 125 by the chloramine T method and purified again by HPLC chromatography.

Un exemple de résultat obtenu avec un anti-sérum de souris immunisée avec le conjugué N17K-SMCC-BSA est présenté à la figure 1 : à 100 µl d'anti-sérum de souris dilué au 1/1000 en tampon PBS-BSA 0,1 %, pH 7,2, on ajoute 50 µl de traceur N17K-SMCC-GT radiomarqué, dans le même tampon et 50 µl de peptide N17K ou de P25 HIV-1 BRU (Transgène) ou de P25 après traitement par la trypsine, à différentes concentrations dans du plasma humain normal. Les complexes antigène-anticorps sont précipités après une incubation de 12 heures à 4° par ajout de 200 µl de PEG 6000 à 20 % dans l'eau et centrifugation. La radioactivité du culot est alors mesurée. Comme le montre la figure 1, les anticorps reconnaissent aussi bien le peptide N17K que la protéine P25 traitée par la trypsine mais pas la protéine P25 native.An example of the result obtained with a mouse anti-serum immunized with the N17K-SMCC-BSA conjugate is presented in FIG. 1: to 100 μl of mouse anti-serum diluted to 1/1000 in PBS-BSA 0 buffer, 1%, pH 7.2, 50 μl of radiolabelled N17K-SMCC-GT tracer are added in the same buffer and 50 μl of N17K peptide or P25 HIV-1 BRU (Transgene) or P25 after treatment with trypsin, at different concentrations in normal human plasma. The antigen-antibody complexes are precipitated after an incubation of 12 hours at 4 ° by adding 200 μl of PEG 6000 at 20% in water and centrifugation. The radioactivity of the pellet is then measured. As shown in FIG. 1, the antibodies recognize both the N17K peptide and the P25 protein treated with trypsin, but not the native P25 protein.

La détermination quantitative du peptide N17K permet donc de réaliser un dosage de la protéine P25 du virus HIV-1 BRU mais devrait permettre également celui des protéines P25 de toutes les souches de virus HIV qui présentent une homologie de structure avec ce fragment.The quantitative determination of the peptide N17K therefore makes it possible to carry out an assay of the P25 protein of the HIV-1 BRU virus but should also allow that of the P25 proteins of all the strains of HIV virus which have a structural homology with this fragment.

Exemple 2 : Dosage de la LPH (Lipotropine Humaine) EXAMPLE 2 Determination of LPH (Human Lipotropin)

On a développé un dosage immunologique de la LPH en utilisant des anticorps produits contre l'hexapeptide NH2 terminal (E6R) : Glu-Leu-Thr-Gly-Gln-Arg.An immunological assay for LPH was developed using antibodies produced against the terminal NH2 hexapeptide (E6R): Glu-Leu-Thr-Gly-Gln-Arg.

Ce peptide qui correspond à un fragment trypsique ne présente aucune homologie significative avec l'ensemble des séquences protéiques contenues dans les banques de données NBRF et GENPRO.This peptide which corresponds to a tryptic fragment has no significant homology with all the protein sequences contained in the NBRF and GENPRO databases.

Dans le but d'obtenir des anticorps dirigés contre les extrémités COOH et NH2 de ce fragment, les immunisations ont été pratiquées avec un peptide dont la séquence est la suivante : Glu-Leu-Thr-Cys-Gln-Arg, où une cystéine remplace la glycine présente dans le fragment originel, afin de pouvoir coupler ce peptide à une protéine porteuse par l'intermédiaire du groupement -SH de cette cystéine.In order to obtain antibodies directed against the COOH and NH2 ends of this fragment, the immunizations were carried out with a peptide whose sequence is as follows: Glu-Leu-Thr-Cys-Gln-Arg, where a cysteine replaces the glycine present in the original fragment, in order to be able to couple this peptide to a carrier protein via the -SH group of this cysteine.

Ce couplage est réalisé par l'intermédiaire de succinimidyl 4 (N-maléimido-méthyl) cyclohexane-1 carboxylate (SMCC) qui réagit, d'une part avec un groupement SH d'une cystéine naturelle ou surnuméraire du peptide et, d'autre part, avec les groupements epsilon NH2 des lysines de la protéine porteuse :This coupling is carried out via succinimidyl 4 (N-maleimido-methyl) cyclohexane-1 carboxylate (SMCC) which reacts, on the one hand with an SH group of a natural or supernumerary cysteine of the peptide and, on the other hand share, with the epsilon NH2 groups of the lysines of the carrier protein:

A de la sérum albumine bovine (Serva) (ci-après BSA), en solution à 5 mg/ml dans un tampon phosphate 0,1 M, pH = 7, on ajoute du SMCC (Pierce) en solution à 25 mg/ml dans le DMF dans un rapport molaire de 1/50. Après une incubation d'une heure à température ambiante, les différents réactifs sont séparés par tamisage moléculaire sur une colonne de Biogel® P2 (Bio-Rad®) équilibrée en tampon phosphate 0,1 M, pH = 6. Le peptide est ensuite ajouté à la solution de BSA-SMCC, dans un rapport molaire de 30/1 et le mélange réactionnel est incubé 12 heures à 4°C. La purification de l'immunogène est réalisée par tamisage moléculaire sur une colonne de Biogel® P4 (Bio-Rad®), équilibrée en solution tampon phosphate (PBS), pH = 7,2.To bovine serum albumin (Serva) (hereinafter BSA), in solution at 5 mg / ml in 0.1 M phosphate buffer, pH = 7, SMCC (Pierce) in solution at 25 mg / ml is added. in DMF in a molar ratio of 1/50. After an incubation of one hour at room temperature, the different reagents are separated by molecular sieving on a column of Biogel® P2 (Bio-Rad®) balanced in 0.1 M phosphate buffer, pH = 6. The peptide is then added to the BSA-SMCC solution, in a molar ratio of 30/1 and the reaction mixture is incubated for 12 hours at 4 ° C. The purification of the immunogen is carried out by molecular sieving on a column of Biogel® P4 (Bio-Rad®), equilibrated in phosphate buffer solution (PBS), pH = 7.2.

Huit souris BALB/c et deux lapins ont été immunisés à intervalle mensuel avec respectivement 50 et 250 µg de cet immunogène, injecté par voie sous-cutanée en adjuvant complet de Freund.Eight BALB / c mice and two rabbits were immunized at monthly intervals with respectively 50 and 250 μg of this immunogen, injected subcutaneously as a complete Freund's adjuvant.

Deux de ces animaux ont produit des anticorps permettant de doser spécifiquement la LPH. Après traitement de l'échantillon sérique par la trypsine qui provoque la libération du peptide NH2 terminal, le dosage est réalisé par compétition avec un peptide identique couplé à glycyl-L-tyrosine par l'intermédiaire de SMCC, radiomarqué à l'iode 125 (figure 2).Two of these animals produced antibodies making it possible to specifically measure LPH. After treatment of the serum sample with trypsin which causes the release of the terminal NH2 peptide, the assay is carried out by competition with an identical peptide coupled to glycyl-L-tyrosine via SMCC, radiolabelled with iodine 125 ( figure 2).

On dispose donc, dans cette application de la présente invention, d'un procédé de dosage immunologique de la LPH, pour laquelle il est particulièrement difficile d'obtenir une quantité suffisante de protéine de pureté convenable pour réaliser des immunisations.Thus, in this application of the present invention, there is a method for the immunological determination of LPH, for which it is particularly difficult to obtain a sufficient quantity of protein of suitable purity to carry out immunizations.

Claims (16)

  1. Process for the identification or determination of a protein in which;
    - after study of its sequence, a peptide which can be liberated by proteolytic enzymatic treatment is selected in the protein,
    - a synthetic peptide having the sequence of the above peptide is prepared by synthesis,
    - antibodies against this synthetic peptide are prepared,
    - a sample optionally containing said protein is subjected to said enzymatic treatment in order to cleave said protein,
    - an immunological determination of the presence of the releasable peptide is proceeded with in said sample, by putting in contact said antibodies with said sample,
    - if desired, the quantity of protein present in the sample is deduced.
  2. Process according to claim 1, characterized in that the peptide contains 6 to 30 amino acids
  3. Process according to claim 1 or 2, characterized in that the peptide contains 6 to 20 amino acids
  4. Process according to any one of claims 1, 2 or 3, characterized in that the peptide corresponds to a part of the C-terminal, N-terminal or median fragment.
  5. Process according to any one of claims 1 to 4, characterized in that the selected peptide is modified.
  6. Process according to any one of claims 1 to 5, characterized in that the peptide is modified by substitution of one or more amino acids.
  7. Process according to any one of claims 1 to 5, characterized in that the peptide is modified by the addition of one or more amino acids.
  8. Process according to any one of claims 1 to 5, characterized in that the peptide is modified by the deletion of one or more amino acids.
  9. Process according to any one of claims 1 to 8, characterized in that the selected peptide can be found in the sequence of a protein, but does not exist naturally in the state of a fragment.
  10. Process according to any one of claims 1 to 9, characterized in that the peptide is selected for its specificity for a single protein.
  11. Process according to any one of claims 1 to 9, characterized in that the peptide is selected for its specificity for a family of proteins.
  12. Process according to any one of claims 1 to 11, characterized in that the peptide is found in polymer form.
  13. Process according to any one of claims 1 to 12 applied to the identification of a protein.
  14. Process according to any one of claims 1 to 12 applied to the determination of a protein.
  15. Process according to any one of claims 1 to 14, characterized in that the protein is the protein P25 of an HIV virus.
  16. Process according to any one of claims 1 to 14, characterized in that the protein is Human Lipotropin (LPH).
EP91430007A 1990-04-06 1991-04-04 Method for identifying or determining proteins and applications therefor Expired - Lifetime EP0451070B1 (en)

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DE10054055A1 (en) 2000-10-31 2002-05-23 Nmi Univ Tuebingen Methods for analyzing proteins
US20030092008A1 (en) * 2001-11-14 2003-05-15 Bell Michael L. Method and apparatus for multiplex flow cytometry analysis of diverse mixed analytes from bodily fluid samples
US7460960B2 (en) * 2002-05-10 2008-12-02 Epitome Biosystems, Inc. Proteome epitope tags and methods of use thereof in protein modification analysis
CA2485560A1 (en) * 2002-05-10 2004-06-03 Engeneos, Inc. Unique recognition sequences and methods of use thereof in protein analysis
US7618788B2 (en) * 2002-05-10 2009-11-17 Millipore Corporation Proteome epitope tags and methods of use thereof in protein modification analysis
US20060014212A1 (en) * 2002-05-10 2006-01-19 Epitome Biosystems, Inc. Proteome epitope tags and methods of use thereof in protein modification analysis
US20050255491A1 (en) * 2003-11-13 2005-11-17 Lee Frank D Small molecule and peptide arrays and uses thereof
US7855057B2 (en) * 2006-03-23 2010-12-21 Millipore Corporation Protein splice variant/isoform discrimination and quantitative measurements thereof
CA2793959C (en) 2010-03-25 2019-06-04 Oregon Health & Science University Cmv glycoproteins and recombinant vectors
PT2691530T (en) 2011-06-10 2018-05-10 Univ Oregon Health & Science Cmv glycoproteins and recombinant vectors
AU2012216792A1 (en) 2011-09-12 2013-03-28 International Aids Vaccine Initiative Immunoselection of recombinant vesicular stomatitis virus expressing HIV-1 proteins by broadly neutralizing antibodies
US9402894B2 (en) 2011-10-27 2016-08-02 International Aids Vaccine Initiative Viral particles derived from an enveloped virus
ES2631608T3 (en) 2012-06-27 2017-09-01 International Aids Vaccine Initiative Env-glycoprotein variant of HIV-1
US20150065381A1 (en) 2013-09-05 2015-03-05 International Aids Vaccine Initiative Methods of identifying novel hiv-1 immunogens
EP2873423B1 (en) 2013-10-07 2017-05-31 International Aids Vaccine Initiative Soluble hiv-1 envelope glycoprotein trimers
US10174292B2 (en) 2015-03-20 2019-01-08 International Aids Vaccine Initiative Soluble HIV-1 envelope glycoprotein trimers
EP3072901A1 (en) 2015-03-23 2016-09-28 International Aids Vaccine Initiative Soluble hiv-1 envelope glycoprotein trimers

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FR2660757A1 (en) 1991-10-11
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